Given the role of the peptides of the invention in modulating the activity of the cellular protein phosphatase 2A, it is important to mention the present state of the art on protein phosphatases 2A, their physiological role and their interactions with some cellular, viral or parasitic proteins.
Cell physiology is controlled in part by modulation of the phosphorylation state of proteins. The phosphorylation state of cellular proteins depends on the antagonistic action of the protein kinases that phosphorylate them and the protein phosphatases that dephosphorylate them.
Protein phosphatases are divided into two main groups: tyrosine phosphatases and serine/threonine phosphatases. Serine/threonine phosphatases are classified into two categories depending on the specificity of their substrate and their sensitivity to certain inhibitors, the type 1 and type 2 phosphatases (PP1 and PP2). The type 2 phosphatases are again divided into different classes, including phosphatase 2A (PP2A), phosphatase 2B (PP2B) or calcineurin which activity is regulated by calcium, and phosphatase 2C (PP2C) which activity is magnesium-dependent.
In vivo, the serine/threonine protein phosphatases PP1 and PP2A form two families of many ubiquitously expressed holoenzymes. These holoenzymes are produced by the specific interaction between their catalytic subunits (PP1c and PP2Ac) and a wide variety of regulatory subunits. Moreover, these holoenzymes are involved in targeting and/or regulation of phosphatase activity (for recent review, see Garcia A. et al., PP1 et PP2A, des ser/thr phosphatases au coeur de l'apoptose (2001) Med/Sci 17, 1214-1216).
It is now known that type 2A phosphatases are very conserved throughout evolution and are potentially activated during regulation of various biological processes. The PP2A enzymes have clearly been involved in transcription control, cell cycle control, and viral transformation. Moreover, the PP2As are the targets of different viral or parasitic proteins, thus suggesting a role for PP2As in host-pathogen interactions.
The PP2As are oligomeric complexes (holoenzymes), each of said complexes comprising a catalytic subunit C and one or two regulatory subunits, (A) and (B). The structure of subunit (A) consists of 15 imperfect repeats of a conserved 38 to 40-amino acid sequence, some subunits (A) interacting with subunits (B) and (C). Subunits (A) and (C), conserved throughout evolution, form the base structure of the enzyme and are constitutively expressed. By contrast, subunits (B) form a family of regulatory proteins differentially expressed and with no common structure between one another (Cohen P. The structure and regulation of protein phosphatases. Annu. Rev. Biochem. 1989; 58:453-508). Therefore, the protein phosphatases 2A are present in vivo under two different forms: a dimeric form (AC) and a trimeric form (ABC). Subunits (B) regulate the phosphatase activity and the specificity towards the substrate. The existence of multiple forms of PP2A correlates with distinct and varying functions of the PP2As in vivo.
Recently, it has been found that different non cellular proteins, and in particular viral and parasitic proteins, are involved in the modulation of some specific activities of protein phosphatases 2A.
Different strategies involving the PP2A are adopted by the viruses to facilitate their replication and survival in the host cell. For example, the parainfluenza virus incorporates, in its viral particle, the PKC ζ protein, a protein of cellular origin under the control of the PP2A. This virus can thus perturb the host proteins' phosphorylation and facilitate its own replication (B. P. Gupta et al. Cellular protein kinase C ζ regulates human parainfluenza virus type 3 replication. Proc. Natl. Acad. Sci. USA 1995; 92:5204-8).
Many DNA viruses with a transforming potential, such as papovaviridae or adenoviruses, as well as some retroviruses, such as the type 1 human immuno-deficiency virus (HIV-1), code for proteins that interact directly with some host PP2As. All these viruses include proteins that, even though they are structurally different from one another, interact with some holoenzymes and modify the phosphatase activity thereof.
More particularly, the E4orf4 protein of adenoviruses binds to a heterotrimeric PP2A and, more precisely, to a regulatory subunit (B), thus leading to a decrease in transcription of JunB in the infected cell. This effect could play an important role during viral infection by regulating the apoptotic response in infected cells. Interestingly, the interaction between E4orf4 and PP2A induces apoptosis of transformed cells in a p53-independent manner (Shtrichman R. et al. Adenovirus type 5 E4 open reading frame 4 protein induces apoptosis in transformed cells. J. Virol. 1998; 72:2975-82).
The oncogenic DNA viruses of the Papovaridae family, including SV40 and the polyoma virus, induce cell transformation. PP2A interacts with the “small T” antigens of SV40 and the “small T” and “middle T” of the polyoma virus. The interactions between these viral proteins and the PP2A are clearly involved in viral transformation. Finally, the transcriptional control, a process normally led in the cell by the different factors which specifically bind to promoter regulatory sequences, represents probably the most important mechanism in viral expression control by PP2A. Therefore, PP2A is a negative regulator of numerous transcription factors, namely involved in the cell growth and proliferation processes, including AP1/SRE, NF-kB, Sp1 and CREB (Waszinski, B. E. et al. Nuclear protein phosphatase 2A dephosphorylates protein kinase A-phosphorylated CREB and regulates CREB Transcriptional stimulation. Mol. Cell Biol. 1993:13, 2822-34). The viral control of these transcription factors could allow to modulate viral transcription.
Vpr, the viral protein of HIV-1, interacts in vitro with PP2A and stimulates the catalytic activity thereof (Tung L, et al. Direct activation of protein phosphatase 2A0 by HIV-1 encoded protein complex Ncp7:vpr. FEBS Lett 1997; 401: 1997-201). Vpr can induce a G2 arrest in infected cells by inhibiting the activation of the p34cdc2-cyclin B complex. In addition, Vpr interacts with the Sp1 transcription factor and is a weak trans-activator of the Sp1-dependent transcription of HIV-1. Therefore, the Vpr protein of HIV-1, which is incorporated within the virion, would be involved in vivo in the initiation of viral transcription, an essential step in regulating the expression of the Tat transcription factor (a major regulator in the transcription coded by the HIV-1 virus).
Unlike the protein kinases that have a well-established role in parasitic infections, serine/threonine phosphatases have only recently been recognized as potentially important regulators in parasitology.
The absence of common motifs for the bulk of proteins that interact with PP2A impedes the simple identification by bio-informatics of peptide motifs directly involved in the binding of these proteins with PP2A.
However, given the major role of protein phosphatases 2A in virus-host or parasite-host interactions as detailed hereinabove, it will be understood that there is a great interest in identifying the binding sites of viral or parasitic proteins with PP2A holoenzymes or one of their subunits, to identify new therapeutic targets for these viral or parasitic pathogens.
The type 1 and type 2A serine/threonine phosphatases (PP1 and PP2A) represent new potentially important targets for apoptotic control, namely in cancer cells, as well as for the control of viral or parasitic infections (for review, see A. Garcia et al. (2000) Protein Phosphatase 2A: a definite player in Viral and parasitic regulation. Microbes Inf. 2,401-407; Et X. Cayla et al. (2000). La Protéine Phosphatase 2A: une nouvelle piste pour l'étude des virus et des parasites. Méd/Sci 16, 122-127). More particularly, PP1/PP2A would play a crucial role in the regulation of anti-apoptotic Bcl-2 proteins and cell survival (Garcia A et al., PP1 et PP2A des ser/thr phosphatases au coeurde l'apoptose (2001). Med/Sci 17, 1214-1416; Ayllón, V. et al. (2000). Protein phophatase 1- is a Ras-activated Bad phosphatase that regulates IL-2 deprivation-induced apoptosis. EMBO J. 19, 2237-2246, Ayllón, V. et al. (2000) Bcl-2 targets protein phosphatase 1 alpha to Bad. J. Immunol. 15; 166:7345-7352). Identification of peptides that interact with PP2A could help to produce new drugs susceptible to block, by competitive inhibition, cellular mechanisms induced by viral or parasitic proteins, by their interaction with PP2A and, in particular, the mechanisms of infection, pathogen proliferation and neoplastic cell transformation.
In particular, PP2A activation after interaction with the E4orf4 adenoviral protein induces apoptosis in transformed cells (Shtrichman R, et al. (2000) Oncogene. 19, 3757-3765). This specific effect requires an interaction with the B alpha (Bα) subunit of PP2A (Marcellus et al. J Virol. (2000) 74:7869-7877)/Goedert et al. J.Neurochem (2000) 75,2155-2162)). All of the above-mentioned observations suggest the hypothesis that the interaction of peptides mimicking the ABC1 and/or A3 site with PP2A could lead to apoptosis of transformed cells.
WO9801563 and WO0104329 A1 describe the human E4orf4 protein and its role in induction of apoptosis in tumour cells, particularly when this protein is expressed via an adenoviral vector. WO0104629 A1 relates to the modulating and mimetic polypeptides E4orf4 and PP2A, capable to induce a selective cell death. This document discloses an invention which relates to the ability of the E4orf4 adenoviral protein to induce the death of neoplastic cells but not of non neoplastic cells. Moreover, WO9801563 A2 relates to the E4orf4 and E4orf6 adenoviral proteins, destined to induce cell death. Finally, the patent application No. FR 0110139 describes peptidic compounds that can bind to PP2A.
Also, the Bcl-2 protein family which, in mammals, comprises about twenty members, can be divided into three sub-families including:                the anti-apoptotic members (of the Bcl-2 type itself) which all present at least four conserved motifs, called “BH1 to BH4” for “Bcl-2 Homology domain”, are necessary to the function of cell survival. The BH4 motif comprises the interaction domain with the Raf and Apaf-1 proteins, and with calcineurine;        the pro-apoptotic members of the Bax type do not present a BH4 domain; and        the pro-apoptotic members of the Bad type only present one BH3 domain.        
Mutagenesis experiments show that the anti-apoptotic activity of Bcl-2 requires its phosphorylation at the specific residue serine 70 that, when replaced by alanine, inhibits survival. Moreover, the work from Dr. May's team (USA) (2001, Vol. 15, No. 4, pp 515-522) suggests that, in the presence of IL-3, PP2A can transitorily associate itself with Bcl-2. The use of a point mutant (wherein one alanine residue replaces the serine 70) indicates that the binding of PP2A to Bcl-2 requires the presence of serine 70 which, consequently, could belong to the binding site. The authors thus suggest a dynamic regulatory mechanism whereby PP2A would be a Bcl-2 phosphatase, antagonistic to Bcl-2 kinases, for example the PKCs (for discussion, see Garcia A. et al., PP1 et PP2A des ser/thr phosphatases au Coeurde I'apoptaose (2001). Med/Sci 17, 1214-1216).
The presence of these Bcl-2 peptides inside the cell could thus regulate the phosphorylation, consequently the activity, of Bcl-2 which in turn could block the development of Bcl-2-dependent tumours.
There is thus a need, at the level of antitumour, antiviral and antiparasitic treatments, for peptides derived from E4orf4 and Bcl-2 sequences which bind to PP2A or one of its subunits.